EP1341754A1

EP1341754A1 - Substituted anthranilic acids

Info

Publication number: EP1341754A1
Application number: EP01994713A
Authority: EP
Inventors: Andreas Weichert; Hans-Willi Jansen; Heinz-Werner Kleemann; Hans-Jochen Lang; Hartmut Rütten
Original assignee: Aventis Pharma Deutschland GmbH
Current assignee: Sanofi Aventis Deutschland GmbH
Priority date: 2000-12-07
Filing date: 2001-11-24
Publication date: 2003-09-10
Anticipated expiration: 2021-11-24
Also published as: EP1341754B1; US20020123632A1; DE50104794D1; JP2004525088A; ES2232679T3; MXPA03005022A; CA2436870A1; JP3978135B2; WO2002046148A1; US6593352B2; DE10060809A1; AR031639A1; AU2002224884A1

Abstract

The invention relates to anthranilic acids of formula (I) wherein the substituents R1-R3 have the designations cited in the claims. Said acids have no unwanted and detrimental salidiuretic properties, but rather very good cardioprotective properties, for example in terms of oxygen deficiency phenomena. Due to their pharmacological properties, said acids are highly suitable as cardioprotective pharmaceuticals for the prophylaxis and treatment of infarction, as well as the treatment of angina pectoris, also preventively inhibiting or strongly reducing the pathophysiological processes in the case of disorders caused by ischaemia. The protective action of the inventive compounds of formula (I) against pathological, hypoxic and ischaemic situations, and the inhibition of the cellular Na<+>/HCO3 cotransporter (NBC), enables said compounds to be used as pharmaceuticals for treating all acute or chronic disorders caused by ischaemia, or diseases induced in a primary or secondary manner by said disorders.

Description

SUBSTITUTED ANTHRANILIC ACIDS

Substituted anthranilic acids, their use as medication or diagnostic, as well as medicament containing them, as well as a pharmaceutical combination preparation with a sodium / hydrogen exchange (NHE) blocker

The invention relates to anthranilic acids of the formula I.

in which mean:

R (1) H, Cl, Br, I, CN, (C <[-Cβ) - alkyl, (C ₃ -C ₆ ) - cycloalkyl or phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents from the group F, Cl, (C-1-C3) - alkyl, methoxy or

- (CF ₂ ) a-CF ₃ ; a zero, 1, 2 or 3;

R (2) (C ^< | -C ₈ ) alkyl, -C _b H _2b - (C3-C ₆ ) - cycloalkyl, -CH ₂ b-phenyl, -C _b H ₂ - pyridinyl, -CbH b-thiophenyl , -CbH ₂ b- furanyl, where the aromatic systems are unsubstituted or substituted with 1-3 substituents from the group F, Cl, CFß, (C-ι-C3) alkyl, methoxy or -SO ₂ NR (4) R (5);

R (4) and R (5) independently of one another H, (C- | -C4) -alkyl, b zero, 1, 2, 3 or 4;

R (3) -C _d H _2d - phenyl, wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents from the group F, Cl, CF3, (C1-C3) - alkyl or

methoxy; d 3 or 4; and their pharmaceutically acceptable salts.

Preferred compounds of the formula I are those in which: R (1) Cl, (C1-C4) alkyl or phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl,

CF3, (C-1-C3) alkyl or methoxy;

R (2) (C1-C4) - alkyl, -CH ₂ b- cyclohexyl, -CbH ₂ b- phenyl, -CbH b- pyridinyl, - CbH ₂ b- thiophenyl, -CbH ₂ ~ furanyl, the aromatic systems being unsubstituted or are substituted with 1 to 3 substituents selected from the group consisting of

F, Cl, CF ₃ , (-C-C ₃ ) alkyl, methoxy or -SO ₂ NH ₂ ; b zero, 1 or 2;

R (3) -n-C4HS-phenyl,

wherein the phenyl is unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF3, (C1-C3) - alkyl or

methoxy; and their pharmaceutically acceptable salts.

Compounds of the formula I are particularly preferred in which: R (1) Cl, (C1-C4) - alkyl or phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF3, (C1-C3) alkyl or methoxy; R (2) (C-1-C4) - alkyl, -CbH ₂ - cyclohexyl, -CbH - phenyl, -CbH ₂ - pyridinyl, - CbH _b - thiophenyl, -CH ₂ b- furanyl, where the aromatic systems are unsubstituted or are substituted with 1-3 substituents selected from the group consisting of F, Cl, CF ₃ , (C1-C3) - alkyl, methoxy or -SO ₂ NH ₂ ; b 1;

R (3) -n-C4H8-phenyl,

and their pharmaceutically acceptable salts.

The connection is very particularly preferred

4- ChIoro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid, and its pharmaceutically acceptable salts.

If one of the substituents R (1) to R (5) contains one or more centers of asymmetry, then both S and R can be configured. The compounds can exist as optical isomers, as diastereomers, as racemates or as mixtures thereof.

The designated alkyl radicals can be either straight-chain or branched. Compounds of the formula I can be synthesized by the person skilled in the art using methods known from the literature.

HLN. III

~ R3

Fluorine or chlorine can be considered as possible escape groups X2 (formula VI) in the nucleophilic aromatic substitution reaction with amines III.

The introduction of some substituents in the 4-position of intermediate II (X1 is bromine, iodine or -O-SO ₂ CF3 is also possible by methods of the literature which are known from the literature

Palladium-mediated cross-couplings of aryl halides or aryl triflates with, for example, organostannans, organoboronic acids or organoboranes or organocopper or zinc compounds.

Anthranilic acids I are generally weak acids that bind bases to form salts. All pharmacologically acceptable salts are suitable as base addition products, for example alkali metal salts, lysinates and tris (hydroxymethyl) methylamine salts.

The compounds I are substituted anthranilic acids. A prominent representative of the anthranilic acid class is the furfuryl derivative furosemide, which is used as a diuretic in therapy. Furosemide inhibits the sodium / potassium / 2chlorocotransporter in the ascending branch of the Henleschen loop in the kidney.

furosemide

DE 18 02 208 describes structurally similar anthranilic acids which, however, carry only benzyl, furfuryl or thienyl substituents on R3 and have diuretic activity. US Pat. No. 3,565,920 claims anthranilic acids which are structurally related to the compounds of the formula I, but are also notable for a strong salidiuretic activity.

It was therefore surprising that the compounds according to the invention have no undesirable and disadvantageous salidiuretic, but very good cardioprotective properties, for example in the case of oxygen deficiency symptoms. As a result of their pharmacological properties, the compounds are outstandingly suitable as cardioprotective medicaments for the prevention of infarction and the treatment of the infarct, as well as for the treatment of angina pectoris, while also preventively inhibiting or greatly reducing the pathophysiological processes when ischemically induced damage occurs. Because of their protective effects against pathological hypoxic and ischemic situations, the compounds of the formula I according to the invention can, as a result of inhibition of the cellular

Na ⁺ / HCθ3 "cotransporters (NBC) as a medicament for the treatment of all acute or chronic damage caused by ischemia or thereby primarily or secondary induced diseases can be used. This affects their use as medicines for surgery, e.g. B. in organ transplants, the compounds can be used both for protecting the organs in the donor before and during removal, for protecting removed organs, for example during treatment with or their storage in physiological bath fluids, as well as for the transfer into the recipient organism , The compounds are also valuable, protective drugs when performing angioplasty surgery, for example on the heart as well as on peripheral vessels. In addition, the compounds according to the invention reduce the development or the extent of heart failure after various insults. Corresponding to their protective action against ischemically induced damage, the compounds are also suitable as medicaments for the treatment of ischemia of the nervous system, in particular of the CNS, whereby they are suitable, for example, for the treatment of stroke or himedema. In addition, the compounds of formula I according to the invention are also suitable for the treatment of forms of shock, such as, for example, allergic, cardiogenic, hypovolemic and bacterial shock.

In addition, the compounds of the formula I according to the invention are notable for a strong inhibitory effect on the proliferation of cells, for example fibroblast cell proliferation and the proliferation of smooth vascular muscle cells. The compounds of the formula I are therefore useful therapeutic agents for diseases in which cell proliferation is a primary or secondary cause and can therefore be used as anti-atherosclerotic agents, agents for late diabetic complications,

Cancer diseases, fibrotic diseases such as pulmonary fibrosis, liver fibrosis or kidney fibrosis, organ hypertrophies and hyperplasias, in particular in prostate hyperplasia or prostate hypertrophy can be used.

The invention further relates to a combination of an NBC blocker of the formula I with sodium / hydrogen exchange (NHE) inhibitors. Surprisingly, both classes of active ingredient show in the combined therapeutic application synergistic effects in the treatment of clinical pictures that are due to ischemic conditions and reperfusion events. Thus, the combinations of an NBC inhibitor with an NHE blocker are outstandingly suitable for the prevention of infarct and infarct and the treatment of infarct, as well as for the treatment of angina pectoris and the inhibition of ischemically induced cardiac arrhythmias, tachycardia and the development and maintenance of ventricular fibrillation, the combinations also preventively inhibit or greatly reduce the pathophysiological processes when ischemically induced damage occurs. Because of their enhanced protective effects against pathological hypoxic and ischemic situations, the inventive

Combinations due to increased inhibition of Na ⁺ influx into the cell can be used as a medicament for the treatment of all acute or chronic damage caused by ischemia or diseases which are primarily or secondarily induced. This affects their use as medicines for surgery, e.g. B. in organ transplants, the combinations of an NHE inhibitor with a blocker of the non-inactivating sodium channel both for the protection of the organs in the donor before and during removal, for the protection of removed organs, for example also in their storage in physiological bath fluids, as well as the transfer into the recipient organism can be used. The combinations of an NBC blocker of the formula I with NHE inhibitors are also valuable, protective drugs when performing angioplasty surgical interventions, for example on the heart and on peripheral vessels. Corresponding to their protective effect against ischemically induced damage, these combinations are also used as medicaments for the treatment of ischemia of the nervous system, in particular the

Central nervous system suitable, where they are suitable for the treatment of stroke or cerebral edema. In addition, the combinations according to the invention are also suitable for the treatment of forms of shock, such as allergic, cardiogenic, hypovolemic and bacterial shock. For example, it was surprisingly found that the combination of the NBC blocker 4-chloro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenyIbutylamino-benzoic acid with the NHE inhibitor cariporide mesilate,

which is described, for example, in US Pat. No. 5,591,754, showed greater than additive, cardioprotective activity in an ischemia / reperfusion model (isolated rat heart).

Medicaments containing a compound I can be administered orally, parenterally, intravenously, rectally or by inhalation, the preferred application depending on the particular appearance of the disease. The compounds I can be used alone or together with pharmaceutical auxiliaries, both in veterinary and in human medicine.

The person skilled in the art is familiar with the auxiliaries which are suitable for the desired pharmaceutical formulation on the basis of his specialist knowledge. In addition to solvents, gel formers, suppository bases, tablet excipients and other active substance carriers, for example antioxidants, dispersants, emulsifiers, defoamers, taste correctives, preservatives, solubilizers or colorants can be used.

For an oral form of use, the active compounds are mixed with the suitable additives, such as carriers, stabilizers or inert diluents, and brought into suitable dosage forms, such as tablets, coated tablets, capsules, aqueous, alcoholic or oily solutions. As inert carriers such. As gum arabic, magnesia, magnesium carbonate, potassium phosphate, milk sugar, glucose or starch, especially corn starch, can be used. The preparation can take place both as dry and as moist granules. Vegetable or animal oils, such as sunflower oil or cod liver oil, are suitable as oily carriers or as solvents.

For subcutaneous or intravenous administration, the active compounds are brought into solution, suspension or emulsion, if desired with the usual substances such as solubilizers, emulsifiers or other auxiliaries. As solvents such. B. in question: water, physiological saline or alcohols, e.g. As ethanol, propanol, glycerol, and also sugar solutions such as glucose or mannitol solutions, or a mixture of the various solvents mentioned.

Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for. B. solutions, suspensions or emulsions of the active ingredient of formula I in a pharmaceutically acceptable solvent, such as in particular ethanol or water, or a mixture of such solvents.

If required, the formulation can also contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers and a propellant. Such a preparation usually contains the active ingredient in a concentration of approximately 0.1 to 10, in particular approximately 0.3 to 3% by weight.

The dosage of the active ingredient of formula I to be administered and the frequency of administration depend on the potency and duration of action of the compounds used; also on the type and severity of the disease to be treated and on the sex, age, weight and individual responsiveness of the mammal to be treated. On average, the daily dose of a compound of the formula I in an approximately 75 kg patient is at least 0.001 mg / kg, preferably 0.01 mg / kg, to at most 10 mg / kg, preferably 1 mg / kg body weight. In acute outbreaks of the disease, such as immediately after suffering a heart attack, even higher and, above all, more frequent doses may be necessary, e.g. B. up to 4 single doses per day. In particular when used intravenously, for example in an infarct patient in the intensive care unit, up to 200 mg per day may be necessary. 0

Experimental part List of abbreviations:

ACN Acetonitrile 5 HPLC High Pressure Liqiud Chromatography

LM solvent

RT room temperature

EE ethyl acetate (EtOAc)

Mp melting point 0 eq. equivalent to

TFA trifluoroacetic acid

Rt retention time

MS mass spectrometer dppf 1, 1'-bis (diphenylphosphino) ferrocene ^

analytics:

HPLC Lagilent 1100 Method gradient: 10% ACN (0.1% TFA) / 90% water (0.1% TFA) - up

- 90% ACN (0.1% TFA) / 10% water (0.1% TFA) in 7 min, flow 2.5 mL / min column: Altech RP18, 3 μm, 7 x 35 mm

MS: Waters Mass Lynx - ESI-TOF, [MH ⁺ ] -, 100% peak unless otherwise stated

HPLC 2Agilent 1100 LC / MSD

Method gradient: 5% ACN (0.05% TFA) / 95% water (0.05% TFA) - up

- 95% ACN (0.05% TFA) / 5% water (0.05% TFA) in 4 min, flow 0.5 mL / min

Column: ESI, [M + H ⁺ ] ⁺ , Merck Purospher RP18, 5 μm, 2 x 55 mm

General instructions for the production of anthranilic acids (I / II)

Level 1: 1.0 eq. The 2,4-bis-halo-5-chlorosulfonyl-benzoic acid of the formula VI is dissolved or suspended in ethyl acetate (5 ml / mmol) and then 5 eq. Amine of formula V. After stirring for 17 hours at RT, the reaction mixture is acidified to pH 1 to 2 with 2N hydrochloric acid and extracted with EA. After drying over MgS04, the LM is evaporated and the crude product is used in the next step without further purification.

Level 2: 1.0 eq. the 2,4-bis-halo-5-sulfamoyl-benzoic acid derivative of the formula IV is 5 eq. Amine of formula III added and heated at 100 ° C for 24 hours. After the reaction mixture has cooled, 5N citric acid solution is added and the mixture is extracted with EA. The organic phase is concentrated and the crude product obtained is purified by preparative HPLC (RP gel, mobile phase acetonitrile / water gradient). Example 1: 4-chloro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid,

Lysine salt, HPLC2: Rt = 5,252 MS: 525.20 Synthesis route: a) 4-Chloro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-chloro-benzoic acid from reaction of 4-chloro-5- (chlorosulfonyl ) -2-chloro-benzoic acid and 4-fluoro-3-chloro-benzylamine according to general instructions, step 1. b) 4-chloro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless crystals. c) Salt formation with 1 eq 1 b), dissolved in acetonitrile, with the addition of an aqueous solution of 1 eq D, L-lysine. After freeze-drying, a colorless solid remains.

Example 2: 4-Bromo-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid,

HPLC2: Rt = 5,270 MS: 569.00

Synthesis route: a) 4-Bromo-5- (chlorosulfonyl) -2-chloro-benzoic acid from 4-bromo-2-chloro-benzoic acid by reaction in pure chlorosulfonic acid (10 eq) at 95 ° C within 6 hours. After cooling, it is poured onto ice and the precipitate is filtered off, a colorless solid. b) 4-Bromo-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-chloro-benzoic acid from 2a) according to general instructions, step 1. c) 4-Bromo-5- (3-chloro-4 -fluorobenzylsulfamoyl) -2-phenylbutylamino-benzoic acid from b) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid. Example 3: 5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid,

HPLC2: Rt = 5,227 MS: 491.40 Synthesis route: a) 5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid from 2c) by hydrogenolysis using a 10% PD / C catalyst in ethanol at RT 2 days. The catalyst is filtered off and the LM is stripped off. The cleaning is carried out by preparative RP-HPLC (water / acetonitrile gradient), colorless solid after freeze-drying.

Example 4: 4-methyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid, HPLC2: Rt = 5,199 MS: 505.05

Synthesis route: a) 4-Bromo-2-fluoro-benzoic acid methyl ester from 4-bromo-2-fluoro-benzoic acid by esterification with an excess of acetyl chloride in methanol at RT within 7 hours. Aqueous work-up and subsequent freeze-drying provide a colorless solid. b) Methyl 4-methyl-2-fluoro-benzoate from 1 eq 4 a) by reaction with 2 eq methyl zinc chloride, prepared from the corresponding Grignard reagent in THF and zinc chloride-THF complex, in the presence of 5 mol% Pd (dppf ) CI ₂ and 6mol%

Cul in THF at RT within 18 h. After NH4CI work-up, the mixture is extracted with EA, the LM evaporated, the crude product via prep. HPLC cleaned and lyophilized, colorless solid. c) 4-methyl-2-fluoro-benzoic acid from 4 b) by hydrolysis with 2N sodium hydroxide solution in methanol at 50 ° C. within one hour. Subsequent acidification with 2N Hydrochloric acid, extraction with EA and drying over MgS04 results in a crude product which is used in the next stage. d) 4-Methyl-5- (chlorosulfonyl) -2-fluoro-benzoic acid from 4 c) by reaction in pure

Chlorosulfonic acid (10 eq) at 95 ° C within 6h. After cooling, it is poured onto ice, extracted with EA and dried over MgSO4. Evaporation produces a yellowish oil, which is further implemented in this purity. e) 4-methyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-fluoro-benzoic acid from 4 d) and 4-fluoro-3-chloro-benzylamine analogous to 1 a), colorless solid.

f) 4-methyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid from 4 e) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 5: 4-isopropyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid,

HPLC2: Rt = 5,399 MS: 533.10

Synthesis route: a) Methyl 4-isopropyl-2-fluoro-benzoate from 1 eq 4 a) by reaction with 2 eq isopropylzinc chloride, analogous to the preparation of 4 a), colorless solid. b) 4-isopropyl-2-fluoro-benzoic acid from 5 a) by hydrolysis with 2N sodium hydroxide solution, analogously to 4 c). c) 4-Isopropyl-5- (chlorosulfonyl) -2-fluoro-benzoic acid from 5 b) by reaction in pure chlorosulfonic acid analogously to 4 d). d) 4-isopropyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-fluoro-benzoic acid from 5 c) and 4-fluoro-3-chloro-benzylamine analogous to 1 a), colorless solid. e) 4-isopropyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid from 5 d) by reaction with phenylbutylamine according to general instructions,

Level 2, colorless solid.

Example 6: 4-n-propyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid,

HPLC2: Rt = 5,437 MS: 533.10

Synthesis route: a) 4- n-propyl-2-fluoro-benzoic acid methyl ester from 1 eq 4 a) by reaction with 2 eq n-propyl zinc chloride, analogous to the representation of 4a), colorless solid. b) 4- n-propyl-2-fluoro-benzoic acid from 6 a) by hydrolysis with 2N sodium hydroxide solution, analogously to 4 c). c) 4- n-propyl-5- (chlorosulfonyl) -2-fluoro-benzoic acid from 6 b) by reaction in pure chlorosulfonic acid analogously to 4 d). d) 4- n-propyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-fluoro-benzoic acid from 6 c) and 4-fluoro-3-chloro-benzylamine analogous to 1 a), colorless solid. e) 4- n-propyl-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylaminobenzoic acid from 6 d) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 7: 4- (4-methylphenyl) -5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid HPLC2: Rt = 5,762 MS: 581.60 from reaction of 1 eq 4-bromo-5 - (3-Chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid and 1.2 eq 4-tolyl-boronic acid in an LM mixture of toluene / MeOH 2: 1 in the presence of 10 mol% Pd (OAc) ₂ , 20 mol% Triphenylphosphine and 3 eq sodium carbonate under reflux for 3 hours.

Aqueous work-up, subsequent preparative HPLC and freeze drying provide a colorless solid.

Example 8: 4-Chloro-2-phenylbutylamino-5 - [(pyridin-4-ylmethyl) sulfamoyl] benzoic acid,

HPLC1: Rt = 4,417 MS: 474.1 [M + H +] +

Synthesis route: a) 2,4-dichloro-5 - [(pyridin-4-ylmethyl) sulfamoyl] benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and pyridin-4-yl-methylamine general procedure, stage 1. b) 4-chloro-2-phenylbutylamino-5 - [(pyridin-4-ylmethyl) sulfamoyl] -benzoic acid from a) by reaction with phenylbutylamine according to general procedure, step 2, colorless solid.

Example 9: 4-Chloro-2-phenylbutylamino-5 - [(pyridin-2-ylmethyl) sulfamoyl] benzoic acid HPLC1: Rt = 4,441 MS: 474.1 [M + H +] +

Synthetic route: a) 2,4-dichloro-5 - [(pyridin-2-ylmethyI) sulfamoyl] benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and pyridin-2-yl-methylamine General regulation, level 1.

b) 4-Chloro-2-phenylbutylamino-5 - [(pyridin-2-ylmethyl) sulfamoyl] benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid. Example 10: 4-Chloro-2-phenylbutylamino-5- (3-chloro-benzylsulfamoyl) -benzoic acid HPLC1: Rt = 5,267 MS: 505.08 Synthetic route: a) 2,4-dichloro-5- (3-chloro-4-fluoro -benzylsulfamoyl) -benzoic acid from the reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and 3-chloro-benzylamine according to general instructions, stage 1. b) 4-chloro-2-phenylbutylamino-5- (3- chloro-benzylsulfamoyl) -benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 11: 4-Chloro-2-phenylbutylamino-5- (4-sulfamoyl-benzylsulfamoyl) benzoic acid HPLC1: MS: 550.13

Synthesis route: a) 4-chloro-5- (4-sulfamoyl-benzylsulfamoyl) -benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and 4-sulfamoyl-benzylamine according to general instructions, stage 1. b ) 4-Chloro-2-phenylbutylamino-5- (4-sulfamoyl-benzylsulfamoyl) -benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 12: 4-Chloro-2-phenylbutylamino-5- (2,3-dichloro-benzylsulfamoyl) benzoic acid

HPLC1: Rt = 5,368 MS: 539.06 Synthesis route: a) 4-chloro-5- (2,3-dichloro-benzylsulfamoyl) -benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and 2,3-dichloro-benzylamine according to general instructions, Stage 1. b) 4-Chloro-2-phenylbutylamino-5- (2,3-dichloro-benzylsulfamoyl) benzoic acid from a) by reaction with phenylbutylamine according to the general procedure, stage 2, colorless solid.

Example 13: 4-Chloro-2-phenylbutylamino-5- (2,4-dichloro-benzy.sulfamoyl) benzoic acid

HPLC1: Rt = 5,433 MS: 539.06

Synthesis route: a) 4-chloro-5- (2,4-dichloro-benzylsulfamoyl) -benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and 2,4-dichloro-benzylamine according to general instructions, Stage 1. b) 4-Chloro-2-phenylbutylamino-5- (2,4-dichloro-benzylsulfamoyl) benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 14: 4-Chloro-2-phenylbutylamino-5- (2-chloro-6-fluoro-benzylsulfamoyl) benzoic acid

HPLC1: Rt = 5,230 MS: 523.11 Synthetic route: a) 4-Ch! Oro-5- (2-chloro-6-fluoro-benzylsulfamoyl) -benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and 2-Chloro-6-fluoro-benzylamine according to general regulations, level 1. b) 4-Chloro-2-phenylbutylamino-5- (2-chloro-6-fluoro-benzylsulfamoyl) -benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless

Solid.

Example 15: 4-Chloro-5 - [(5-methyl-furan-2-ylmethyl) sulfamoyl] -2-phenylbutylamino-benzoic acid

HPLC1: Rt = 5,036 MS: 513.19

Synthetic route: a) 2,4-dichloro-5 - [(5-methyl-furan-2-ylmethyl) sulfamoyl] benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and 5-methyl- furan-2-yl-methylamine according to general instructions, stage 1. b) 4-chloro-5 - [(5-methyl-furan-2-ylmethyl) sulfamoyl] -2-phenylbutylamino-benzoic acid from a) by reaction with Phenylbutylamine according to general instructions, level 2, colorless solid.

Example 16: 4-Chloro-5- [2- (4-chlorophenyl) ethylsulfamoyl] -2-phenylbutylaminobenzoic acid HPLC1: Rt = 5,453 MS: 519.14

Synthetic route: a) 2,4-dichloro-5- [2- (4-chlorophenyl) ethylsulfamoyl] benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and 2- (4-chloro -phenyl) -ethylamine according to general instructions, stage 1. b) 4-chloro-5- [2- (4-chlorophenyl) ethylsulfamoyl] -2-phenylbutylamino-benzoic acid from a) by reaction with phenylbutylamine according to general. Regulation, level 2, colorless solid. Example 17: 4-Chloro-2-phenylbutylamino-5- (2-thiophene-2-yl-ethylsulfamoyl) benzoic acid

HPLC1: Rt = 5,253 MS: 491.10 Synthetic route: a) 2,4-dichloro-5- (2-thiophene-2-yl-ethylsulfamoyl) benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and 2-thiophene-2-yl-ethylamine according to general instructions, stage 1.

b) 4-Chloro-2-phenylbutylamino-5- (2-thiophene-2-yl-ethylsulfamoyl) benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 18: 4-Chloro-5- (4-ethyl-phenylsulfamoyl) -2-phenylbutylamino-benzoic acid

HPLC1: Rt = 5,358 MS: 485.17

Synthetic route: a) 2,4-dichloro-5- (4-ethyl-phenylsulfamoyl) benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and 4-ethyl-aniline according to general instructions, stage 1 b) 4-chloro-5- (4-ethyl-phenylsulfamoyl) -2-phenylbutylamino-benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 19: 4-Chloro-5- (phenylbutylsulfamoyl) -2-phenylbutylamino-benzoic acid HPLC1: Rt = 5,511 MS: 523.11 from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid with phenylbutylamine according to the general procedure, stage 2, colorless solid.

Example 20: 4-Chloro-5- (cyclohexylmethylsulfamoyl) -2-phenylbutylaminobenzoic acid

HPLC1: Rt = 5,548 MS: 475.10

Synthetic route: a) 2,4-dichloro-5- (cyclohexylmethylsulfamoyl) benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) benzoic acid and cyclohexylamine according to general instructions,

Stage 1. b) 4-Chloro-5- (cyclohexylmethylsulfamoyl) -2-phenylbutylamino-benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Example 21: 4-Chloro-5- (isobutylsulfamoyl) -2-phenylbutylamino-benzoic acid

HPLC1: Rt = 5,208 MS: 437.10

Synthesis route: a) 2,4-dichloro-5- (isobutyl-sulfamoyl) -benzoic acid from reaction of 2,4-dichloro-5- (chlorosulfonyl) -benzoic acid and isobutylamine according to general instructions, stage 1. b) 4-chloro -5- (isobutyl-sulfamoyl) -2-phenylbutylamino-benzoic acid from a) by reaction with phenylbutylamine according to general instructions, stage 2, colorless solid.

Pharmacological part:

Description of NBC activity measurements: Most of the molecular biological techniques follow protocols from the works "Current Protocols in Molecular Biology (eds. Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA and Struhl, K .; John Wiley & Sons) "or:" Molecular Cloning: A Laboratory Manual (Sambrook, J., Fritsch, EF and Maniatis, T .; Cold Spring Harbor Laboratory Press (1989)) ". First, the heart shape of the human NBC1 was cloned by RT-PCR. After confirmation of the sequence, the corresponding cDNAs were cloned into the vector pcDNA3.1 +, which contains the neo gene as a selection marker for eukaryotic cells. For the amplification of the human heart shape of the NBC1, human heart mRNA (from Clontech, Palo Alto, CA, USA) was amplified with primers which cover the area in which the heart shape differs from the kidney shape. The heart shape differs only at the 5 'end of the coding sequence. The region coding for the first 41 amino acids of the kidney shape is replaced in the heart shape by a region coding for 85 amino acids (positions 118-370 from Abukadze et al., J. Biol. Chem. 273, 17689-17695 (1998) or positions 45-294 from Choi et al., Am. J. Physiol. Cell Physiol. 276, C576-C584 (1999) replace positions 150-270 from Burnham et al. (See above), J. Biol. Chem. 272, 19111 - 19 114 (1997) The product of the PCR reaction was first cloned into the vector pCR2.1 and, after verification of the sequence, was cloned into the cDNA of the kidney form of the NBC1 by means of interfaces introduced by the PCR reaction Sequencing for correct incorporation of the human heart NBC1 cDNA checked. The plasmids obtained for the heart shape of the human NBC1 were transfected into the cell line CHO K1 (Chinese hamster ovary cells) using the LipofectAmine ™ reagent from LifeTechnologies (Gaithersburg, MD, USA). di e has no measurable NBC activity. After selection for transfected cells by growth in G418-containing medium (only cells which have received a neo gene by transfection can survive under these conditions), individual cells were isolated and cultured. With the test described below, cell clones were identified on the FLIPR that have a clear NBC activity. The best cell lines were used for the further tests and cultivated to avoid loss of the transfected sequence under constant selection pressure in medium containing G418. In order to determine the inhibitory activity of the active substances on the heart shape of human NBC1, a test was set up which developed a further development for the

Testing of inhibitors of the NCBE (Na ⁺ -dependent CI7HCO3 exchanger) (EP 0 903 339) based on the "acid load" method (Sardet et al., Cell 56, 271-280 (1989); Faber et al ., Cell. Physiol. Biochem. 6, 39-49 (1996). In this test the recovery of the intracellular pH (pHj) after a previous acidification is determined under conditions in which the NBC is active, the other pHj regulating systems of CHO cells like NHE (Na ⁺ / H ⁺ -

Exchanger) and NCBE (Na ⁺ -dependent CI HC03_ exchanger) are blocked by specific inhibitors. This ensures that the observed intracellular pH recovery is due to the activity of NBC1.

Carrying out the measurements

The day before, the transfected cells are sown on 96-well microtiter plates with a density of approx. 15,000 cells / well in 200 μl growth medium and incubated overnight at 37 ° C. in the CO ₂ incubator. The intracellular pH of the transfected

Cells with the pH-sensitive fluorescent dye BCECF (Molecular Probes (Eugene, OR, USA), the precursor BCECF-AM) is used. The cells are first loaded with BCECF-AM. The growth medium of the cells sown the day before is removed manually since the fetal calf serum present in the medium could interfere with the BCECF staining. For staining, 100 μl of NH4CI staining buffer (20 mM NH4CI, 115 mM NaCI, 1 mM

MgSO4, 1mM CaCl2, 5mM KCI, 20mM HEPES, 5mM Glucose; pH adjusted to 7.4 with 1 M NaOH), which contains 5 μM BCECF-AM. The cells are incubated with this staining solution for 20 minutes at 37 ° C. in a CO 2 incubator. During this period, on the one hand NH4 ⁺ ions accumulate in the cells, which causes a slight alkalization, on the other hand BCECF-AM gets into the cells, where the BCECF dye, which is not permeable to the cell membrane, from BCECF-AM comes into play through the action of esterases is released. To acidify the Cells are then washed thoroughly in the cell washer (three times with a total volume of 1, 2 ml per well) with a Na ⁺ - and NH4 ⁺ -free

Wash buffer washed (133.8 mM choline chloride, 4.7 mM KCI, 1, 25 mM CaCl2,

1.25 mM MgCl2, 0.97 mM K2HPO4, 0.23 mM KH2PO4, 5 mM HEPES, 5 mM glucose; pH adjusted to 7.4 with 1 M KOH). This washing ride leads to a drastic drop in the intracellular pH (-6.3-6.4). However, since the washing buffer contains neither sodium nor bicarbonate ions, the cells are unable to regulate their intracellular pH. The actual measurement of the intracellular pH recovery takes place in the so-called FLIPR (Fluorescence Imaging Plate Reader) from Molecular Devices (Sunnyvale, CA, USA). The FLIPR has one

Argon laser, whose 488 nm band is very suitable for excitation of the BCECF. Due to the complicated beam guidance, all 96 wells of a microtiter plate can be excited simultaneously and thus measured simultaneously. The special design of the FLIPR only excites the lower 50 μm in each well, which is why it is preferred to use adherent ones

Cells such as CHO works. The light emitted by the excited cells first passes through a filter that is transparent between 510 and 570 nm, and is then registered using a CCD camera. Since the FLIPR also contains a built-in 96-tip pipettor, the same volume of any liquid can be pipetted into all 96 wells of a microtiter plate at the same time. A complete measurement of a complete microtiter plate can be carried out approximately every second. In the FLIPR, 180 μl of substance buffer (90 mM NaCl, 25 mM NaHCO 3, 25 mM KCI, 1 mM MgCl2, 1 mM CaCl2, 0.8 mM K2HPO4, 0.2 mM KH2PO4, 10 mM HEPES) are added to the cells acidified after washing , 5 mM glucose; pure CO _{2 is} passed through for 5 seconds on the day of the measurement and the pH is then adjusted to 7.4 with 1 M NaOH; however, the CO ₂ passing through can also be omitted without the measurement results changing appreciably). In order to inhibit the pH regulation systems NHE and NCBE, which are also active under these buffer conditions, the substance buffer additionally contains specific inhibitors of these exchangers. The final concentration of the NHE inhibitor cariporide mesilat (EP 589 336) is 10 μM, that of the NBCE inhibitor according to EP 855 392 Example 1: 2-Butyl-5-methylsulfanyl-3- (2'-cyanaminosulfonyl-biphenyl-4-ylmethyl) -3H-imidazole-4-carboxylic acid ethyl ester 30 μM. After adding substance buffer, the intracellular pH of the previously acidified cells increases due to the activity of the NBC, which is reflected in an increase in the fluorescence of the pH-sensitive dye.

The inhibitory effect of a substance is now determined by comparing the fluorescence increase, which is linear with the pH increase, under the influence of this substance with that of wells in which the NBC is uninhibited [100% activity, only addition of Cariporide mesilat and of the NCBE blocker according to EP 855 392. Example 1] or is completely inhibited [0% activity, in addition to Cariporide mesilate and NCBE blocker according to EP 855 392, Example 1, and addition of 400 μM DCDPC, 4-chloro-2 - (3-chloro-phenylamino) benzoic acid].

The entire measurement of a microtiter plate takes two minutes, with the entire plate being measured every 2 seconds. After the first 5 measurements, the 180 μl substance buffer containing the compounds to be tested are pipetted to the acidified cells at a rate of 60 μl per second. After a few seconds, wells in which the NBC is not inhibited show a clear increase in fluorescence. The range between 20 and 80 seconds in which the fluorescence increase in the positive controls is linear is considered to calculate the remaining NBC activity. Of the 96 wells of the microtiter plate, 8 are used for the 100% and 0% values.

The following data refer to the residual activity at an inhibitor concentration of 10μM and are the result of double determinations. , Results: [%] at 10μM

Example 1 46

2 56

3 88

4 54

5 78

6 69

7 95

8 89

9 89

10 75

11 81

12 37

13 60

14 87

15 79

16 91

17 95

18 79

19 77

20 89

21 94

Claims

claims

1. Anthranilic acids of the formula

in which mean:

R (1) H, Cl, Br, I, CN, (Ci-Cβ) - alkyl, (C ₃ -C ₆ ) - cycloalkyl or phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents from the group F, Cl, (C1-C3) - alkyl, methoxy or

- (CF ₂ ) a-CF ₃ ; a zero, 1, 2 or 3;

R (2) (C <| -C ₈ ) - alkyl, -C _b H _2b - (C ₃ -C ₆ ) - cycloalkyl, -C _b H _2b - phenyl, -C _b H _2b -

Pyridinyl, -CH - thiophenyl, -CH - furanyl, where the aromatic systems are unsubstituted or substituted with 1-3 substituents from the group F, Cl, CF3, (-C-C3) alkyl, methoxy or -SO ₂ NR (4th ) R (5);

R (3) -Cc (H _c | - phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents from the group F, Cl, CF3, (C-1-C3) - alkyl or

methoxy; d 3 or 4; and their pharmaceutically acceptable salts.

2. Compounds of the formula I according to claim 1, in which: R (1) Cl, (Ci-C4) alkyl or phenyl,

wherein the aromatic nucleus is unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF3, (C1-C3) alkyl or methoxy;

R (2) (C-1-C4) - alkyl, -C _b H _b - cyclohexyl, -C _b H _b - phenyl, -CH _2b - pyridinyl, -

C _b H _2b - thiophenyl, -C _b H _2b - furanyl, where the aromatic systems are unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF3, (-C-C3) alkyl, methoxy or -SO ₂ NH ₂ ; b zero, 1 or 2;

R (3) -n-C4H «- phenyl,

wherein the phenyl is unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF3, (C-J-C3) alkyl or methoxy; and their pharmaceutically acceptable salts.

3. Compounds of formula I according to claim 1, characterized in that therein: R (1) CI, (C1 -C4) - alkyl or phenyl;

R (2) (C1-C4) - alkyl, -C _b H ₂ - cyclohexyl, -C _b H _b - phenyl, -C _b H _2b - pyridinyl, - C _b H _2b - thiophenyl, -C _b H _2b - furanyl; wherein the aromatic systems are unsubstituted or substituted with 1-3 substituents selected from the group consisting of F, Cl, CF ₃ , (C-1-C3) alkyl, methoxy or -SO ₂ NH ₂ ; b 1;

R (3) -n-C4HS-phenyl,

and their pharmaceutically acceptable salts.

4. A compound according to claim 1, characterized in that it is 4-chloro-5- (3-chloro-4-fluoro-benzylsulfamoyl) -2-phenylbutylamino-benzoic acid; as well as their pharmaceutically acceptable salts.

5. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment of arrhythmias.

6. Method for treating arrhythmias, characterized in that an effective amount of a compound I according to claim 1 is mixed with the usual additives and administered in a suitable dosage form.

7. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of heart attack.

8. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of angina pectoris.

9. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of ischemic conditions of the heart.

10. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of ischemic conditions of the peripheral and central nervous system and stroke.

11. Use of a compound I according to claim 1 for the preparation of a

Medicament for the treatment or prophylaxis of ischemic conditions of peripheral organs and limbs.

12. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment of shock conditions.

13. Use of a compound I according to claim 1 for the manufacture of a medicament for use in surgical operations and organ transplants.

14. Use of a compound I according to claim 1 for the manufacture of a medicament for the preservation and storage of grafts for surgical measures.

15. Use of a compound I according to claim I for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause

16. Use of a compound I according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of disorders of the fat metabolism.

17. Medicament containing an effective amount of a compound I according to one or more of claims 1 to 4.